Project Summary/Abstract RNA modification enzymes are ubiquitous among the three domains of life and necessary for the proper function of many cellular RNAs. RNA substrates may be unstructured or highly base-paired as in tRNA or rRNA. Direct inspection of the sequence of highly base-paired RNAs is challenging because of the deep and narrow major groove of the RNA helix. How RNA modification enzymes achieve specificity for highly base- paired substrates is an open question. Two mechanisms feature prominently: protein promoted melting of base pairs to form interactions between the protein and the Watson-Crick faces of nucleotides or protein recognition of a specific three-dimensional RNA shape indirectly driven by RNA sequence. It is previously unknown how general these mechanisms are among RNA modification enzymes and if a few paradigms exist which can explain the specificity mechanisms of many classes of RNA modification enzymes. This proposal will use characterization of the structure and mechanism of the erythromycin resistance methyltransferase enzyme family to address the problem of RNA modification enzyme specificity, moving toward the goal of identifying common mechanistic strategies used for specific modification of highly base-paired RNA substrates. Erythromycin resistance methyltransferase enzymes methylate a specific adenosine residue of rRNA adjacent to the peptidyl transferase center of the ribosome sterically occluding the binding of multiple antibiotics that target the ribosome. The enzymes contribute to the significant public health problem of antibiotic resistance and also are an excellent model system for basic science. In Aims 1 and 2, steady-state and pre-steady state kinetics assays will be performed to understand how protein structure and RNA sequence and structure drive specificity. Wild type protein and rRNA substrates will be used along with site-directed mutants of both enzyme and rRNA towards the goal of building a kinetic mechanism for RNA methylation and understanding how specific protein-RNA interactions contribute to the mechanism. Aim 3 encompasses experiments using small- angle x-ray scattering to build a three-dimensional model of enzyme interaction with rRNA. Throughout the Aims we will assay two evolutionarily distinct members of the erythromycin resistance methyltransferase family to determine if there is an idiosyncratic or conserved mechanism for this enzyme family.